An improved understanding of how B cell responses to the HlV-1 envelope glycoproteins (Env) are regulated is needed to accelerate the development of an HIV-1 vaccine. In this project, we will investigate the B cell repertoire engaged in the initial response to Env and determine how this response evolves over time. Our recently established methodology to conduct basic B cell studies in non-human primates (NHPs) represents a substantial step forward for vaccine research and an opportunity to obtain information about B cell maturation and selection processes that will directly impact the design of improved Env immunogens and the selection of Env immunogens for use in clinical trials. In Aim 1, we will use existing NHP samples from Env trimer immunized macaques to examine if a longer immunization interval stimulates antibodies that display an incresaed level of somatic hypermutation (SHM). We will also examine genetic features ofthe memory B cell repertoire directed against the CD4-binding site (CD4bs) of Env and ask if critical B cell specificities are lost as the response evolves over time. In Aim 2, we will examine B cell responses elicited by new generation soluble Env trimers and isolate monoclonal antibodies for genetic and functional analysis. Specifically, we will ask if the V(D)J gene usage ofthe CD4bs-specific B cell repertoire is altered when immunogens engineered to present the CD4bs in a conformation-constrained manner are used, or in response to sequential prime-boosting with heterologous immunogens, and we will assess the impact of this on the antibody neutralizing activity. In Aim 3, we will investigate the Env-specific repertoire of long-lived plasma cells residing in the bone marrow of immunized rhesus macaques. This aim will include the development of methods to optimize single cell sorting of bone marrow-resident plasma cells. We will ask if the bone marrow repertoire is established early in the immune response and how diverse this population is compared to the Env-specific memory B cell pool. By addressing these questions, we hope to obtain an improved understanding about Env immunogenicity in healthy, primate hosts that is of direct relevance for the design of improved HIV-1 immunogens and the selection of Env candidates for clinical studies.